Newborn screening programs can be effective at saving lives as well as preventing serious morbidity, such as mental retardation, through the early detection and treatment of genetic conditions. Thus, screens for newborns should be expanded to include additional disorders, particularly those having treatment options.
Genetic conditions where a newborn screening program could be particularly useful include those involving alterations in sterol pathways. These conditions can cause untimely mortality and morbidity and include conditions such as cerebrotendinous xanthomatosis (CTX), Smith-Lemli-Opitz syndrome, familial hyper-cholesterolaemia, and congenital adrenal hyperplasia. CTX has been identified as a candidate for universal newborn screening in a recent American College of Medical Genetics report. However, to be effective, screening and/or diagnosis methods for CTX must have sufficient sensitivity and selectivity.
CTX is a rare genetic sterol condition caused by mutations in the CYP27A gene (the CYP27A gene is also known as sterol 27-hydroxylase gene). The CYP27A gene is associated with the P450 enzyme is important in production of bile acids. When CYP27A activity is inhibited or blocked, bile acid precursors will accumulate in various tissues. CTX often presents itself in childhood with the symptoms caused by the accumulation of the ketosterols bile acid precursors and their derivatives slowly increasing. Often CTX is not identified until adulthood after significant damage has been done. The biochemical phenotype of CTX is shown in FIG. 12.
Screening of biomarkers for 27-hydroxylase deficiencies has previously been considered. However, the biomarkers for CTX are ketosterol biomarker compounds and such screening presents a challenge because these biomarkers are generally present at low levels in clinical and biological samples such as plasma. Additionally, the biomarkers may be present at even lower levels in unaffected individuals, making it difficult to obtain a reference. The highly hydrophobic ketosteroids pose chromatographic challenge when using Reversed Phase (RP) chromatography. The detection and quantification of ketosterol biomarker steroids by mass analysis are also particularly challenging because of poor ionization efficiency of such compounds, complex ionization patterns, interference in the mass measurement by isobaric compounds and low sample concentrations in the sample medium.
Standard chromatographic techniques such as GC-MS methods for analysis after chemical derivatization are available. See, e.g., Song, J. et al., Journal of Chromatography B., Vol. 791, Issues 1-2, (127-135) 2003, the contents of which are incorporated by reference. Methods using fluorescence detection and some immunoassays, including radioimmunoassays (RIAs), are also available, but these usually do not offer multi-component analysis. The major problems with RIAs are lack of specificity and the need to perform a different assay for each steroid.
One existing analytical method to analyze ketosterols such as 5α-cholest-3-one and other bile acid precursors that accumulate in CTX and bowel disease has been described, for example, in U.S. Pat. No. 8,158,435, Andrea E. DeBarber, et. al.; Clinica Chimica Acta, 411 (2010) 43-48; Akira Honda, et. al.: The Journal of Biological Chemistry 276, 37 (2001) 34579-34585; and M. Camilleri et. al. Neurogastroenterol Motil (2009) 21,734-e43 Technical Note, herein incorporated by reference. GC-FID or GC-MS was used for the biochemical screening of CTX using Girard P derivatization with ESI-MSMS detection. However, limitations of GC based methodology include a lengthy analysis time (>30 min) and complex sample preparation.
The above challenges associated with detecting and quantifying ketosterol biomarker compounds in samples are magnified by the desire to rapidly screen and/or analyze a large number of biological samples for the specific compounds of interest or a panel of ketosterol bile acid precursors.
Additionally, conventional methods can be labor intensive and lack the requisite sensitivity to measure normal unaffected patient serum or whole blood samples, particularly for newborn screening, where typically only low sample volumes are available. It is hence desirable to have simple and specific methods for analyzing levels of CTX biomarkers in affected and normal human samples.
Thus, there remains a need for suitable sensitive and selective screening tests for identification and quantification of CTX. Such a test is preferably sensitive and specific for quantitation of ketosterols and other analytes that are useful as markers for CTX.